Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
Functions of Bone
The function of bone is to provide mechanical support for joints, tendons and ligaments, to protect vital organs from damage and to act as a reservoir for calcium and phosphate in the preservation of normal mineral homeostasis. Diseases of bone compromise these functions, leading to clinical problems such as fracture, bone pain, bone deformity and abnormalities of calcium and phosphate homeostasis.
Types of Bone
The normal skeleton contains two types of bone; cortical or compact bone, which makes up most of the shafts (diaphysis) of the long bones such as the femur and tibia, and trabecular or spongy bone which makes up most of the vertebral bodies and the ends of the long bones.
All bone is subject to continual turnover, with old bone being actively resorbed, and new bone being deposited. This turnover, or “remodelling” is essential for maintenance of structural competence because continual loading results in the formation of numerous microfractures in the bone matrix which, if left unchecked, would be weak points that could seed catastrophic failures of the bone, i.e., clinically obvious fractures. Such a process can be likened to a stone-chip on an automobile windscreen: the small crack can act as a catalyst for the sudden failure of the entire structure.
Remodelling is therefore an essential process for the maintaining bone strength. As the bone is resorbed and redeposited, the microfractures and structural imperfections are removed.
Trabecular bone has a greater surface area than cortical bone and because of this is remodeled more rapidly. Consequently, conditions associated with increased bone turnover tend to affect trabecular bone more quickly and more profoundly than cortical bone. Cortical bone is arranged in so-called Haversian systems which consists of a series of concentric lamellae of collagen fibres surrounding a central canal that contains blood vessels. Nutrients reach the central parts of the bone by an interconnecting system of canaliculi that run between osteocytes buried deep within bone matrix and lining cells on the bone surface. Trabecular bone has a similar structure, but here the lamellae run in parallel to the bone surface, rather than concentrically as in cortical bone.
Bone Composition
The organic component of bone matrix comprises mainly of type I collagen: a fibrillar protein formed from three protein chains, wound together in a triple helix. Collagen type I is laid down by bone forming cells (osteoblasts) in organised parallel sheets (lamellae). Type I collagen is a member of the collagen superfamily of related proteins which all share the unique structural motif of a left-handed triple helix. The presence of this structural motif, which is responsible for the mechanical strength of collagen sheets, imposes certain absolute requirements on the primary amino acid sequence of the protein. If these requirements are not met, the protein cannot form into the triple helix characteristic of collagens. The most important structural requirements are the presence of glycine amino acid residues at every third position (where the amino acid side chain points in towards the center of the triple helix) and proline residues at every third position to provide both structural rigidity and periodicity on the helix. Glycine is required because it has the smallest side chain of all the proteogenic amino acids (just a single hydrogen atom) and so can be accommodated in the spatially constrained interior of the helix. Proline is required because proline is the only secondary amine among the 20 proteogenic acids, which introduces a rigid ‘bend’ in the polypeptide, such that the presence of proline residues at repeated intervals will result in the adoption of a helical conformation.
After synthesis, the collagen protein is the subject of post-translational modifications which are essential for the structural rigidity required in bone. Firstly, collagen becomes hydroxylated on certain proline and lysine residues (e.g. to form hydoxyproline and hydroxylysine, respectively). This hydroxylation depends on the activity of enzymes that require vitamin C as a cofactor. Vitamin C deficiency leads to scurvy, a disease in which bone and other collagen-containing tissues (such as skin, tendon and connective tissue) are structurally weakened. This demonstrates the essential requirement for normal collagen hydroxylation.
After deposition into the bone, the collagen chains become cross-linked by specialised covalent bonds (pyridinium cross-links) which help to give bone its tensile strength. These cross links are formed by the action of enzymes on the hydroxylated amino acids (particularly hydroxylysine) in the collagen. It is the absence of these crosslinks which results in the weakened state of the tissue in scurvy when hydroxylation is inhibited by the absence of sufficient vitamin C.
The biochemical structure of collagen is an important factor in the strength of bone, but the pattern in which it is laid down is also important. The collagen fibres should be laid down in ordered sheets for maximal tensile strength. However, when bone is formed rapidly (for example in Paget's disease, or in bone metastases), the lamellae are laid down in a disorderly fashion giving rise to “woven bone,” which is mechanically weak and easily fractured.
Bone matrix also contains small amounts of other collagens and several non-collagenous proteins and glycoproteins. The function of non-collagenous bone proteins is unclear, but it is thought that they are involved in mediating the attachment of bone cells to bone matrix, and in regulating bone cell activity during the process of bone remodelling. The organic component of bone forms a framework (called osteoid) upon which mineralisation occurs. After a lag phase of about 10 days, the matrix becomes mineralised, as hydroxyapatite ((Ca10(PO4)6(OH)2) crystals are deposited in the spaces between collagen fibrils. Mineralisation confers upon bone the property of mechanical rigidity, which complements the tensile strength, and elasticity derived from bone collagen.
Bone Cell Function and Bone Remodelling
The mechanical integrity of the skeleton is maintained by the process of bone remodelling, which occurs throughout life, in order that damaged bone can be replaced by new bone. Remodelling can be divided into four phases; resorption; reversal, formation, and quiescence (see, e.g., Raisz, 1988; Mundy, 1996). At any one time approximately 10% of bone surface in the adult skeleton is undergoing active remodelled whereas the remaining 90% is quiescent.
Osteoclast Formation and Differentiation
Remodelling commences with attraction of bone resorbing cells (osteoclasts) to the site, which is to be resorbed. These are multinucleated phagocytic cells, rich in the enzyme tartrate-resistant acid phosphatase, which are formed by fusion of precursors derived from the cells of monocyte/macrophage lineage. Osteoclast formation and activation is dependent on close contact between osteoclast precursors and bone marrow stromal cells. Stromal cells secrete the cytokine M-CSF, which is essential for differentiation of both osteoclasts and macrophages from a common precursor.
Mature osteoclasts form a tight seal over the bone surface and resorb bone by secreting hydrochloric acid and proteolytic enzymes through the “ruffled border” into a space beneath the osteoclast (Howship's lacuna). The hydrochloric acid secreted by osteoclasts dissolves hydroxyapatite and allows proteolytic enzymes (mainly Cathepsin K and matrix metalloproteinases) to degrade collagen and other matrix proteins. Deficiency of these proteins causes osteopetrosis which is a disease associated with increased bone mineral density and osteoclast dysfunction. After resorption is completed osteoclasts undergo programmed cell death (apoptosis), in the so-called reversal phase which heralds the start of bone formation.
Osteoblast Formation and Differentiation
Bone formation begins with attraction of osteoblast precursors, which are derived from mesenchymal stem cells in the bone marrow, to the bone surface. Although these cells have the potential to differentiate into many cell types including adipocytes, myocytes, and chondrocytes, in the bone matrix they are driven towards an osteoblastic fate. Mature osteoblasts are plump cuboidal cells, which are responsible for the production of bone matrix. They are rich in the enzyme alkaline phosphatase and the protein osteocalcin, which are used clinically as serum markers of osteoblast activity. Osteoblasts lay down bone matrix which is initially unmineralised (osteoid), but which subsequently becomes calcified after about 10 days to form mature bone. During bone formation, some osteoblasts become trapped within the matrix and differentiate into osteocytes, whereas others differentiate into flattened “lining cells” which cover the bone surface. Osteocytes connect with one another and with lining cells on the bone surface by an intricate network of cytoplasmic processes, running through cannaliculi in bone matrix. Osteocytes appear to act as sensors of mechanical strain in the skeleton, and release signalling molecules such as prostaglandins and nitric oxide (NO), which modulate the function of neighbouring bone cells.
Regulation of Bone Remodelling
Bone remodelling is a highly organised process, but the mechanisms which determine where and when remodelling occurs are poorly understood. Mechanical stimuli and areas of micro-damage are likely to be important in determining the sites at which remodelling occurs in the normal skeleton. Increased bone remodelling may result from local or systemic release of inflammatory cytokines like interleukin-1 and tumour necrosis factor in inflammatory diseases. Calciotropic hormones such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D, act together to increase bone remodelling on a systemic basis allowing skeletal calcium to be mobilised for maintenance of plasma calcium homeostasis. Bone remodelling is also increased by other hormones such as thyroid hormone and growth hormone, but suppressed by oestrogen, androgens and calcitonin. There has been considerable study of the processes which regulate the bone resorption side of the balance, but the factors regulating the rate of bone deposition are considerably less well understood.
Bone Disorders
There are a range of disorders of bone which result from the failure to properly regulate the metabolic processes which govern bone turnover (e.g., metabolic bone disorders).
Osteoporosis (OP) is the most prevalent metabolic bone disease. It is characterized by reduced bone mineral density (BMD), deterioration of bone tissue, and increased risk of fracture, e.g., of the hip, spine, and wrist. Many factors contribute to the pathogenesis of osteoporosis including poor diet, lack of exercise, smoking, and excessive alcohol intake. Osteoporosis may also arise in association with inflammatory diseases such as rheumatoid arthritis, endocrine diseases such as thyrotoxicosis, and with certain drug treatments such as glucocorticoids. However there is also a strong genetic component in the pathogenesis of osteoporosis.
Osteoporosis is a major health problem in developed countries. As many as 60% of women suffer from osteoporosis, as defined by the World Health Organisation (WHO), with half of these suffers also having clinically relevant skeletal fractures. Thus 1 in 3 of all women in developed countries will have a skeletal fracture due to osteoporosis. This is a major cause of morbidity and mortality leading to massive health care costs (an estimated $14 billion per annum in the USA alone) (see, e.g., Melton et al., 1992).
Osteopetrosis, the opposite of osteoporosis, is characterised by excessive bone mineral density. It is, however, much rarer than osteoporosis with as few as 1 in 25,000 women affected.
After osteoporosis, the next most prevalent bone disease is osteoarthritis. Osteoarthritis (OA) is the most common form of arthritis in adults, with symptomatic disease affecting roughly 10% of the US population over the age of 30 (see, e.g., Felson et al., 1998). Because OA affects the weight bearing joints of the knee and hip more frequently than other joints, osteoarthritis accounts for more physical disability among the elderly than any other disease (see, e.g., Guccione et al., 1994). Osteoarthritis is the most common cause of total knee and hip replacement surgery, and hence offers significant economic as well as quality of life burden. Recent estimates suggest the total cost of osteoarthritis to the economy, accounting for lost working days, early retirement and medical treatment may exceed 2% of the gross domestic product (see, e.g., Yelin, 1998).
The physiological mechanisms which underlie osteoarthritis remain hotly debated (see, e.g., Felson et al., 2000) but it seems certain that several environmental factors contribute, including excess mechanical loading of the joints, acute joint injury, and diet, as well as a strong genetic component. The disease is characterised by the narrowing of the synovial space in the joint, inflammatory and fibrous changes to the connective tissue, and altered turnover of connective tissue proteins, including the primary connective tissue collagen, type II. The most recent studies suggest that osteoarthritis may result from misregulated connective tissue remodelling in much the same way that osteoporosis results from misregulated bone remodelling. Whereas osteoporosis is a disease of quantitatively low bone mineral density, osteoarthritis is a disease of spatially inappropriate bone mineralisation.
There are a range of other less common bone disorders, including:
Ricketts and osteomalacia are the result of vitamin D deficiency. Vitamin D is required for absorption of calcium and phosphate and for their proper incorporation into bone mineral. Deficiency of vitamin D (called Ricketts in children and osteomalacia in adults) results in a range of symptoms including low bone mineral density, bone deformation and in severe cases muscle tetany due to depletion of extracellular calcium ion stores.
Hyperparathyroidism (over production of parathyroid horomone or PTH) can have similar symptoms to Ricketts. This is unsurprising since PTH production is stimulated in Ricketts as an attempt to maintain the free calcium ion concentration. PTH stimulates bone resorption by promoting osteoclast activity, and hence can result in symptoms resembling osteoporosis. Osteomalacia and hyperparathyoidism combined contribute only a very small fraction of all cases of adult osteoporosis. In almost every case, adult osteoporosis is due to defective bone deposition rather than overactive resorption (see, e.g. Guyton, 1991).
Paget's disease of bone is a relatively common condition (affecting as many as 1 in 1000 people in some areas of the world) of unknown cause, characterized by increased bone turnover and disorganized bone remodeling, with areas of increased osteoclastic and osteoblast activity. Although Pagetic bone is often denser than normal bone, the abnormal architecture causes the bone to be mechanically weak, resulting in bone deformity and increased susceptibility to pathological fracture.
Multiple myeloma is a cancer of plasma cells. In contrast to most other haematological malignancies, the tumour cells do not circulate in the blood, but accumulate in the bone marrow where they give rise to high levels of cytokines that activate osteoclastic bone resorption (e.g., interleukin-6). The disease accounts for approximately 20% of all haematological cancers and is mainly a disease of elderly people.
Balance Between Bone Deposition and Bone Resorption
All of the bone pathologies listed above result from an imbalance between bone deposition and bone resorption. If the mechanisms regulating these two processes become uncoupled than pathological changes in bone mineral density result. In just a few cases, the cause of the imbalance seems clear: for example prolonged estrogen deficiency (such as due to surgical sterilisation) or lengthy treatment with glucocorticoids (such as for asthma) both perturb the balance and can lead to rapid demineralisation of the bone and osteoporosis.
Unfortunately, in the vast majority of cases the mechanisms resulting in loss of balance are much less clear. The difficulty in identifying the causes stems in part of the small scale imbalances that must be occurring. For example, most osteoporotic fractures do not occur until 20-30 years after the menopause. If, as is generally assumed, the osteoporosis was initiated by the reduction in estrogen levels after the menopause, then the demineralisation has been occurring steadily over two or three decades. Since the bone remodelling process is relatively rapid (complete within 28 days in any given osteon) we must assume that the imbalance in favour of demineralisation is very small.
Current Treatments
There are currently two major classes of drugs used in the prevention and treatment of osteoporosis: (1) Hormonally active medications (estrogens, selective estrogen receptor modulators (SERMs)); and (2) anti-resorptives.
There is presently good data to suggest that the long term use of hormonally active medications (usually estrogen, estrogen analogs or conjugated estrogens) after the menopause in women can prevent bone demineralisation and hence delay the onset of osteoporosis. The molecular mechanisms involved are not clearly defined, possibly because they are so complex. However, there are plausible mechanisms which involve both stimulation of bone deposition and suppression of resorption.
To date, such hormonally active medications, including the new generation of SERMs, such as Raloxifene™, which have the beneficial effects of estrogen on bone and the cardiovascular system but do not have the side effects of breast and uterine hyperplasia that can increase the risk of cancer, have not achieved widespread use for the treatment of existing osteoporosis.
At present, treatment of known or suspected bone mineral deficiency is most commonly by the use of drugs to suppress osteoclast activity. The two most important drug groups in this class are bisphophonates (BPs) and non-steroidal anti-inflammatory drugs (NSAIDs).
Bisphosphonates (also know as diphosphonates) are an important class of drugs used in the treatment of bone diseases involving excessive bone destruction or resorption, e.g., Paget's disease, tumour-associated osteolysis, and also in post-menopausal osteoporosis where the defect might be in either bone deposition or resorption. Bisphosphonates are structural analogues of naturally occurring pyrophosphate. Whereas pyrophosphate consists of two phosphate groups linked by an oxygen atom (P—O—P), bisphosphonates have two phosphate groups linked by a carbon atom (P—C—P). This makes bisphosphonates very stable and resistant to degradation. Furthermore, like pyrophosphate, bisphosphonates have very high affinity for calcium and therefore target to bone mineral in vivo. The carbon atom that links the two phosphate groups has two side chains attached to it, which can be altered in structure. This gives rise to a multitude of bisphosphonate compounds with different anti-resorptive potencies. Bone resorption is mediated by highly specialised, multinucleated osteoclast cells. Bisphosphonate drugs specifically inhibit the activity and survival of these cells. Firstly, after intravenous or oral administration, the bisphosphonates are rapidly cleared from the circulation and bind to bone mineral. As the mineral is then resorbed and dissolved by osteoclasts, it is thought that the drug is released from the bone mineral and is internalised by osteoclasts. Intracellular accumulation of the drugs inhibits the ability of the cells to resorb bone (probably by interfering with signal transduction pathways or cellular metabolism) and causes osteoclast apoptosis (see, e.g., Hughes et al., 1997).
NSAIDs are widely used in the treatment of inflammatory diseases, but often cause severe gastrointestinal (GI) side effects, due their inhibition of the prostaglandin-generating enzyme, cyclooxygenase (COX). Recently developed selective cyclooxygenase-2 (COX-2) inhibitors offer new treatment strategies which are likely to be less toxic to the GI tract. NSAIDs developed by Nicox SA (Sophia Antipolis, France), that contain a nitric oxide (NO)-donor group (NO-NSAID) exhibit anti-inflammatory properties without causing GI side effects. The mechanisms responsible for the beneficial effects of NSAIDs on bone are not definitively identified, but since the bone resorbing osteoclast cells are derived from the circulating monocyte pool, it is not difficult to imagine why generalised anti-inflammatory treatments might have anti-resoptive effects. However, another class of powerful anti-inflammatory molecules, the glucacorticoids and their analogs such as dexamethasone have the opposite effects to NSAIDs: chronic dexamethasone treatment (for example, in asthma) induces demineralisation and leads to symptoms of rapid onset osteoporosis. Consequently, while NSAIDs empirically have anti-resorptive properties, further investigations into the detail mechanism of action of these drugs are clearly required.
It has recently been discovered that many of the drugs, which are used clinically to inhibit bone resorption, such as bisphosphonates and oestrogen do so by promoting osteoclast apoptosis (see, e.g., Hughes et al., 1997). At present the most commonly used types of drugs used to suppress osteoclast activity in these diseases are bisphophonates (BPs) and non-steroidal anti-inflammatory drugs (NSAIDs).
Limitations of Current Treatments
There are a number of limitations which impact on the clinical utility of all the available therapeutic and preventative modalities. For example, both hormonal medications (HRT and SERMs) and antiresorptives (BPs and NSAIDs) primarily target resorption. While this may be useful in, for example Paget's disease, it is likely to be less useful in osteoporosis, where the majority of cases have reduced deposition rates as the primary defect. Of course, because bone mineral density is a balance between deposition and resorption rates, antiresorptive strategies can have some efficacy even where the primary defect is in the rate of deposition.
Possibly because current therapeutics target resorption when suppressed deposition is the primary defect in osteoporosis, none of the current agents can build bone, but instead only halt further demineralisation. Because of the limited availability of diagnostic techniques, particularly for population screening, treatment cannot usually begin until clinical symptoms exist (such as fracture) by which point the bones may already be dangerously demineralised. In such cases (which are the majority), a therapy which increases bone mineral density would be desirable. A new treatment based on abolishing proline deficiency would stimulate deposition rate and hence be a new category of therapeutic: one which targets deposition preferentially over resorption. Therapeutics of this categoy would be expected to overcome the limitation of being unable to increase bone mineral density.
Another limitation of exisiting therapies is the failure to treat the underlying cause of the pathology, but rather to try and alleviate the symptoms. In part, this is because few direct causes of osteoporosis have been identified. The inventors have identified a novel contributory mechanism to the development of osteoporosis and hence have provided the first therapeutic approach to target one of the direct mechanisms resulting in pathologically low bone mineral density.
Bone Disorder Diagnostics
It has long been clear that early diagnosis of bone disorders was essential for good therapeutic management. Although there are now several effective treatments for osteoporosis, each one is only able to arrest the further loss of bone mineral density. No treatment to date has been effective in reversing loss which has already occurred. Thus early, reliable diagnosis of declining bone mineral density is of the utmost clinical importance.
Existing diagnosis methods for bone disorders fall into two categories:                (a) direct observation (for example, bone mineral density scans for osteoporosis or radiographic assessment for osteoarthritis); and,        (b) indirect observation of molecular markers of remodelling (for example, collagen breakdown products).        
Of the major determinants for bone fracture, only bone mineral density can presently be determined with any precision and accuracy.
Bone densitometers typically give results in absolute terms (i.e., bone mineral density, BMD, typically in units of g/cm2) or in relative terms (T-scores or Z-scores) which are derived from the BMD value. The Z-score compares a patient's BMD result with BMD measurements taken from a suitable control population, which is usually a group of healthy people matched for sex and age, and probably also weight. The T-score compares the patient's BMD result BMD measurements taken from a control population of healthy young adults, matched for sex. In other words, for Z-scores, age- and sex-matched controls are used; for T-scores just sex-matched controls are used. The World Health Organisation (WHO) defines osteoporosis as a bone mineral density (BMD) below a cut-off value which is 1.5 standard deviations (SDs) below the mean value for the age- and sex-matched controls (Z-scores), or a bone mineral density (BMD) below a cut-off value which is 2.5 standard deviations (SDs) below the mean value for the sex-matched controls (T-scores) (see, e.g., World Health Organisation, 1994).
The two most widely used methods for assessing bone mineral density (BMD) is the DEXA scan (dual emission X-ray absorbtion scanning) and ultrasound. The DEXA method is considered the gold standard diagnostic tool for bone mineral density, providing a reliable estimate of average bone mineral density in units of grams per cubic centimetre. It can be applied to a number of different bones, but is most commonly used to measure lumbar spine density (as a measure of cortical bone) and femoral neck density (as a measure of trebecular bone mineral density). Ultrasound is easier and cheaper to perform than DEXA scanning, but provides a less reliable estimate of bone mineral density and its accuracy is compromised by the surrounding soft tissue. As a result, ultrasound is usually performed on the heel, where interference by soft tissue is minimised, but it is unclear whether this is typical of whole body bone mineral density, and in any case it does not allow an assessment of cortical bone. See, for example, Pocock et al., 2000; Prince, 2001.
Almost all of the molecular diagnostics currently employed are based on measurements of bone breakdown products. The steady state level of breakdown products should be related to the bone remodelling rate, although it will be biased towards detection of overactive resorption rather than underactive deposition. It may be, in part, for this reason that all therapies currently on trial for osteoporosis (such as estrogen receptor modulators or bisphosphonates) are based on an antiresorptive strategy rather than on promoting deposition, even though (as noted above) most cases of osteoporosis are not due to overactive resorption.
Examples of molecular diagnostics include the measurement of free crosslinks, hydroxyproline, collagen propeptides, or alkaline phosphatase in serum or urine. Free crosslinks are produced when collagen is degraded during resorption. Although the collagen can mostly be broken down to free amino acids, the trimerised hydroxylysine residues that formed the crosslinks cannot be further metabolised and so accumulate in the blood until secreted by the kidney in urine. Thus the levels of crosslink in serum or in urine will be related to the rate of collagen breakdown (most, but not all, of which will be occurring in the bone). Tests for hydroxyproline rely on a similar principle: free proline (that is, proline not incorporated into protein) is never in the hydroxylated form, hydroxyproline. As a result, the only source of free hydroxyproline in blood is from collagen breakdown. As for crosslinks, the free hydroxyproline generated during breakdown cannot be metabolised any further and accumulates until excreted by the kidney. Unfortunately, the level of both of these metabolites (in either serum or urine) is significantly affected by kidney function.
Collagen is produced as a proprotein which has both an N-terminal and C-terminal extension cleaved off prior to incorporation into the extracellular matrix. These extensions, or propeptides, are then metabolised or excreted. However, the steady state level of the propeptides has been suggested to be a marker for collagen deposition, some, but not all, of which is likely to be occurring in the bone.
Problems with Current Diagnostic Methods
The gold standard bone densitometry method, DEXA scanning, is too cumbersome and expensive for routine screening procedures in women without clinical signs of osteoporosis. It requires specialist apparatus (which is large and expensive to install and maintain) as well as specialist training for its operation. Despite accurately measuring bone mineral density, and hence providing the benchmark diagnosis of osteoporosis, nevertheless it does not accurately predict future fracture risk, suggesting that bone quality as well as density may also be important (see, for example, the comments above).
Ultrasound measurements on the heel are simpler to perform, using cheaper apparatus and requiring less operator training, but the results are generally less able to predict the presence of either osteoporosis or future fracture risk.
Molecular diagnostics are considerably easier to implement, although in many cases the reagents required for the assays are expensive to obtain. The major disadvantage of the markers which have been evaluated to date is that the levels of the breakdown products in serum or urine are not particularly temporally stable, changing with diurnal rhythm and also from day to day. As a result, spot measures (i.e., a single specimen taken at a randomly chosen time) have virtually no diagnostic or prognostic power. Series of measurements can be used to provide some indication of relative risk for osteoporosis, but the odds ratio for having osteoporosis is only approximately 2-fold among individuals with high levels of the turnover markers (see, e.g., Garnero, 1996). Such a weak association is of little or no practical clinical value, and as a result, biochemical markers of bone metabolism have not found widespread application in the conical arena, and have not been considered for population screening.
Another important limitation of current molecular diagnostics is the focus on the products of bone metabolism (such as cross links, hydroxyproline, and collagen propetides). These species might offer diagnostic potential but they provide no information at all about the underlying causes of the imbalance between deposition and resorption. Identification of a risk factor that was not a direct marker of bone turnover may offer the prospect of identifying therapeutic targets as well as having prognostic potential.
Metabonomic methods involve obtaining a high density data set which contains information on the identities and relative amounts of all of the low molecular weight substances in a biologial sample (in the present case, human blood serum, although other biofluids can be used as well as tissue samples). These data sets are subjected to pattern recognition or multivariate statistical analyses to identify metabolites, the presence or relative amounts of which are specifically associated with the sample class (e.g., control vs. patient with a particular disease).
As discussed in detail below, the inventors have applied the technique of metabonomics to osteoporosis and have identified a novel biomarker for bone disorders, for example, conditions associated with low bone mineral density, such as osteoporosis: free proline.
Proline
Proline is an alpha-amino acid and one of the twenty proteogenic amino acids (i.e., one of the twenty amino acids which can be incorporated during de novo protein synthesis). Although proteins can contain amino acids other than the basic set of twenty, this only occurs through post-translation modification (e.g., hydroxylation of proline or lysine, gamma-carboxylation of glutamate, etc.). Since the 1950s, all 20 of the proteogenic amino acids have been known to be present in the free form (i.e., not incorporated into a peptide or protein) in human blood (see, e.g., Stein et al., 1954a, 1954b) at levels between 20 μM and 500 μM. Generally, the levels of the amino acids in blood are tightly regulated and do not vary to a great extent between individuals and as a result they are not routinely measured in clinical studies.
Proline, shown below, is one of several amino acids with an alkyl side chain, but is unique among the proteogenic amino acids in that it is a secondary amine, and a cyclic amine, and is, more precisely, an imino acid. This has important structural consequences when proline is incorporated into a polypeptide, causing the chain to “bend”. Where a particular protein structure, such as a left-handed helix, is required, proline is the only amino acid capable of providing rigidity to such a structural motif. Although proline exists in the D- or L-configuration, the D-configuration is most common in a biological setting. Free proline may be in a non-ionic form or in an ionic form (e.g., as a zwitterion), as is usually the case in solution at physiological pH.

Although present in almost every protein, proline is a particularly important constituent of the extracellular matrix proteins of the collagen family. Proline is important both in terms of function (its secondary amine structure promotes helical rigidity) and also in terms of amount. All collagens are constructed from the repeated tripeptide motif -Gly-X-Pro- where Gly is glycine, X is any amino acid, and Pro is proline. Thus, almost one-third by mass of all fibrillar collagens (such as type I collagen in bone or type II collagen in connective tissue) is made up of proline. No other known protein has a mass fraction of proline even approaching this value.
Unlike many other amino acids (such as glycine, glutamate, and tryptophan), free proline has not been implicated in any metabolic pathways other than peptide and protein synthesis. Glycine and glutamate are directly active as signalling molecules, while tryptophan is widely metabolised into signalling molecules such as serotonin. These amino acids (glycine, glutamate) and amino acid derivatives (serotonin, dopamine, adrenalin, etc.) play essential roles in the nervous system as neurotransmitters and may play other key signalling roles, for example, in the control of the immune system. In contrast, no similar roles have been identified for free proline and no biologically active proline-derived metabolites have been reported. As a result, the biochemistry of free proline is much less well understood than for most of the other proteogenic amino acids.
As a result, there are relatively few metabolic reactions which involve free proline: (a) Synthesis of loaded tRNA-Pro, the first step in the incorporation of proline into peptides and proteins. (b) Reactions involved in the synthesis of proline through interconversion of amino acids. In mammals, there are two such pathways: (i) Synthesis from glutamate which involves the sequential action of the enzymes gamma-glutamyl kinase, gamma-glutamyl phospthate reductase (which are separate activities of the same enzyme) and □-pyrroline-5-carboxylate reductase (P5C-reductase). (ii) Synthesis from arginine, via ornithine, which involves the sequential activity of ornithine transaminase and P5C-reductase. In unicellular organisms, there additional proline-utilising pathways (e.g., proline racemase, D-proline reductase and ornithine cyclase have all be identified in Clostridium species. (c) Reactions involved in the catabolism of proline, for interconversion into other amino acids. In mammals, the enzyme proline oxidase, which converts proline into P5C is the major catabolic enzyme for proline. The resulting P5C can be further metabolised to glutamate or arginine (via ornithine) or it can be converted back to proline by P5C reductase.
The only other enzymes which act on proline do so only when the proline has been incorporated as a peptidyl-prolyl residue in a polypeptide chain. Such enzymes include proline hydroxylase, the vitamin C dependent enzyme necessary for generating crosslinks in collagen; and peptidylproplyl cis-trans isomerase, an enigmatic family of enzymes whose physiological role is poorly defined, but which has been widely studied after it was discovered to be the target of major immunosuppressive drugs such as cyclosporin.
The total body supply of proline (most of which is incorporated into collagen in bone and muscle at any given time) is derived from two sources:                (a) dietary supply (for example, from the hydrolysis of dietary protein); and,        (b) endogenous synthesis (proline is a non-essential amino acid because humans retain the biochemical pathways necessary to synthesise it).        
In order to be taken up from the dietary protein supply, the protein must be efficiently hydrolysed in the stomach, and specific uptake mechanisms then transport the peptides containing proline across the gut epithelium. These small peptides are then subjected to enzymatic hydrolysis to release their free amino acids into the blood.
Proline derived from the diet is supplemented by synthesis, primarily by the liver. The synthesis pathway begins with the citric acid cycle intermediate α-ketoglutarate which is converted into another non-esstential amino acid, glutamate. This glutamate, or glutamate obtained directly from the diet, is then converted via a three step pathway into proline. First, the glutamate is reacted with ATP to form glutamic-γ-semi-aldehyde. This product has two fates: it can either be converted into ornithine and hence to arginine, or else it loses water and is cyclised to form Δ-pyrroline-5-carboxylate. This intermediate is then reduced by the enzyme Δ-pyrolline 5-carboxylate dehydrogenase (P5CDH) to give proline. Alternatively, proline may be synthesised from dietary arginine via ornithine and the enzyme ornithine transaminase, which converts ornithine into Δ-pyrroline-5-carboxylate and thence to proline via the action of P5C reductase. The relative contribution of the two synthetic pathways in not well understood, but the glutamate pathway is likely to be the major contributor under most circumstances.
Free proline in the blood is lost through three routes: (a) incorporation into proteins, mainly collagen; (b) a small amount of renal excretion; and, (c) metabolism to other amino acids, such as arginine and glutamate. The vast majority of the free proline is used to support the high level of collagen turnover in the healthy individual. Renal excretion is very low because proline, unique among the proteogenic amino acids, has a specific re-uptake mechanism in the kidney nephron. The evolution of such a mechanism underlies the value placed on retaining the whole body supply of proline. Specific genetic disorders of this process can lead to hyperprolinuria, and this may in these rare cases result in serum proline deficiency.
To be utilised in protein synthesis, and specifically in collagen biosynthesis, there must not only be a sufficient total body supply of proline, but it must also be available to the cells performing the protein synthesis. Like other small charged molecules, proline is unable to cross the plasma membrane by diffusion, but must be transported. Proline taken up into cells by the System A amino acid transporter responsible for all uptake of all proteogenic amino acids with neutral side chains. The system A transporter has been cloned (it is the product of the SAT2 gene) and is inhibited by the “ideal” subtrate methylaminoisobutyrate (MeAlB). Thus, proline transport (for example across the gut epithelium, or into osteoblasts) may also be an important regulatory step both in the determination of serum proline levels and in the determination of collagen biosynthesis rates. Interestingly, tissues engaged in the highest levels of collagen biosynthesis (e.g., bone) have the highest levels of SAT2 expression, and agents which promote collagen formation (e.g., the cytokine TGF-beta) stimulate SAT2 expression and proline uptake capacity in parallel (see, e.g., Ensenat et al., 2001).
Hydroxyproline, in contrast to free proline, is not used for protein synthesis. It cannot be incorporated directly into protein and must instead be generated by the action of prolyl hydroxylase on polypeptides containing proline. It has no other biological activity ascribed to it, and is essentially a waste product from collagen breakdown. It is plausible that hydroxyproline could interfere with other steps in the proline metabolic pathways (e.g., with the synthesis of proline, by product inhibition of the P5C reductase enzyme, or with the kidney re-uptake mechanism, or the System A amino acid transporter); however, there is presently little evidence to support this hypothesis. Any evidence for such action of hydroxyproline would convert it from the role of innocent bystander in osteoporosis to a potential causal contributor.
Free proline is an important component of the bone turnover cycle because bone remodelling demands by far the highest amounts of free proline of any process in the adult, specifically, for de novo collagen synthesis. It has long been suggested that proline is necessary for collagen synthesis. However, to date, there has been no evidence that proline is rate limiting for bone synthesis.
The inventors have now demonstrated that proline is not only necessary, but is rate limiting for new bone formation. Consequently, sub-optimal levels of available free proline cause osteoporosis by slightly slowing the rate of collagen biosynthesis, and hence tipping the balance slightly in favour of demineralisation over a long time period. Furthermore, the inventors have demonstrated, for the first time, that a low concentration of free proline is a risk factor for osteoporosis.
Low Proline Levels
There are many reasons for low proline levels, and these include:
(1) insufficient dietary intake of proline;
(2) failure to absorb dietary proline, e.g., due to a malabsorption defect.
(3) failure to synthesize proline, e.g., due to an enzymatic/genetic defect.
(4) kidney disorder, e.g., malfunction of selective re-uptake of proline.
The proline content of various diets is likely to differ more markedly than for any other free amino acid. Although the total amount of protein intake varies somewhat between individuals, the most dramatic dietary variations are in the nature of the proteins eaten between vegans, vegetarians, and meat-eaters. Collagens, which have by far the highest proline content per gram of protein, are uniquely found in animals as opposed to plants. As a result, the proline content of a vegetarian diet may be less than 50%, and possibly as low as 20%, of the levels in an average meat-eater diet. Thus, both the overall protein content of the diet and the nature of the protein consumed will have a substantial effect on the total amount of proline available for dietary absorption. Individuals who have low proline levels due to dietary insufficiency would be amenable to therapy with proline supplements, e.g., oral supplements.
Even if the diet is replete with proline, the contribution of dietary sources to the plasma pool of free proline will be inadequate if the available proline is not properly absorbed and processed. Many of these steps are in common with other amino acids (e.g., hydrolysis by stomach acids and enzymes, bulk phase pinocytosis of peptides by gut epithelium, etc.). However, the body may be less sensitive to malabsorption of other amino acids for which the whole body demand is less. Individuals with low proline levels due to malabsorption syndromes will not generally be amenable to therapy with oral proline, but may require parenteral administration of proline or treatment of the underlying cause of the proline malabsorption.
Although dietary sources of proline are likely to be important, based on the rapid increase in serum free proline following an oral proline-rich meal (see, e.g., Stein et al., 1954a, 1954b), endogenous synthesis is also presumably important. By analogy with other systems, such as the cholesterol metabolic pathway, endogenous synthesis is usually regulated to provide additional product only when nutritional sources are inadequate. Thus, dietary insufficiency or malabsorption might reveal an underlying defect in the biosynthesis pathway that normalises free proline levels in healthy individuals. Such a defect might be genetic or epigenetic in origin: for example, polymorphisms may exist in the enzymes involved in proline biosynthesis (e.g., P5C reductase) which operate at slightly different rates, or which are subject to subtly different control mechanisms.
As has already been noted, proline is specifically reabsorbed by the kidney. As a result, any disease with alters kidney function could result in lower free proline levels through loss via the kidneys. Such kidney loss may be very significant, and both dietary and endogenous synthesis pathways may be incapable of normalising free proline levels if proline were lost via the kidneys at a rate comparable to some amino acids (e.g., serine). Such genetic defects resulting in hyperprolinuria have already been described in the literature, although no data on their bone mineral density is yet available.
Also, accumulated hydroxyproline from bone breakdown might interefere with proline absorption, synthesis, cellular transport, or renal re-uptake, resulting in a secondary proline deficiency. Elevated levels of hydroxyproline might arise from increased bone turnover (e.g., in Ricketts or hyperthyroidism) or as a result of failure to clear hydroxyproline through the normal renal excretion mechanism.